The broad objective of this grant proposal is to study the mechanisms responsible for the fidelity of DMA synthesis. DMA polymerases are the key enzymes involved in replication and repair of DMA. An analysis of how polymerases control fidelity is central to understanding the biochemical basis of a wide variety of genetic diseases. Lesch-Nyhan syndrome and ADA deficiency are two examples of inherited childhood diseases that can arise from a single point mutation. Activation of oncogenes and inactivation of tumor suppressor genes leading to cancer can result from single base changes in DNA. Genetic defects in post-replication mismatch repair of DNA polymerase errors are a root cause of hereditary nonpolyposis colin cancer, along with a variety of other types of cancer. Previous fidelity studies have focused on individual DNA polymerases in the absence of polymerase accessory proteins required to sustain processive synthesis. This grant investigates the fidelity of purified procaryotic and eucaryotic DNA polymerase holoenzymes, pol III and pol IIfrom Escherichia coli, and pol delta from Schizosaccharomyces pombe. A thorough understanding of fidelity mechanisms of DNA polymerases requires an analysis of the effects of sequence context and replication assessory proteins on fidelity. The proposed experiments, which include a full complement of polymerase subunits, are among the first of its kind, and make use of a mathematical model of polymerase fidelity and a gel fidelity assay that we've developed previously. The model is used to predict the effect of polymerase processivity subunits on base substitution fidelity. These predictions will be tested experimentally. Steady state kinetic experiments are designed to investigate the biochemical basis of mutational "hot" and "cold" spots. We propose to test the importance of hydrogen bonds between Watson-Crick base pairs on polymerase fidelity, by measuring the effects of base stacking on polymerase fidelity, in the absence of hydrogen bonding. Presteady state kinetic experiments, using fluorescent nucleotide analogs, are proposed to measure switching between polymerase and exonuclease active sites in "real-time". A second set of presteady state experiments are designed to determine the mechanism for loading and unloading the polymerase processivity clamp subunit onto DNA and to analyze the requirements for ATP hydrolysis for each step in the clamp-loading pathway. This pathway is required for Okazaki fragment formation during discontinuous lagging-strand DNA synthesis. PERFORMANCE SIT t(S) (organization, city, state) University of Southern California Department of Biological Sciences University Park Los Angeles, CA 90089-1340 KEY PERSONNEL. See instructions on Page 11. Use continuation pages as needed to provide the required information in the format shown below. Name Organization Role on Project Myron F. Goodman DSC Principal Investigator Phuong T. Pham USC PostDoct. Res. Assoc. Michael J. Hartenstine USC Graduate Res. Assistant Xiluo Chen USC Graduate Res. Assistant Eli Lazarov USC Graduate Res. Assistant Mike O'Donnell Rockefeller Univ. Collaborator Jerard Hurwitz Sloane-Kettering Inst. Collaborator Joe Beechem Vanderbilt Univ. Collaborator Eric Kool Univ. of Rochester Collaborator PHS 398 (Rev. 4/98) Page2 BB Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. CC Principal Investigator/Program Director (Last, first, middle): Goodman, Myron F. Type the name of the principal investigator/prograMdirector at the top of each printed page and each corj^uation page. (For type specifications, see instructions on page 6.) Bl RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,